Congenital Diaphragmatic Hernia (CDH) is a frequent and often fatal developmental condition of diaphragm defects associated with lung hypoplasia caused by diverse factors that we hypothesize are predominantly genetic, but heterogeneous. Project IV will use a combination of clinical, molecular, developmental, genomic and cytogenetic strategies to identify mutations causing CDH, with the hope of elucidating perturbed biochemical pathways that can then serve as functional targets for pharmacological intervention or treatment. Aim I: We will recruit a cohort of carefully phenotyped isolated and complex CDH patients from two major clinical sites, MGH and Children's Hospital Boston, establish cell lines on each patient, and parents,and siblings, and enter deceased populations of patients, families with multiple affected members, consanguineous families, and monozygotic twins. DNA will be extracted from various sources for mutational analysis of candidate genes, arrayed based Comparative Genomic Hybridization (aCGH), loss of heterozygosity studies (LOH), and metaphase preparations for subtelomeric FISH. Aim II: High resolution cytogenetic tools such as 1 Mb array CDH, subtelomeric FISH, and multiplex ligand-directed probe amplification (MLPA) will be used to identify microdeletions, microduplications, and balanced and unbalanced translocations followed by breakpoint analyses to identify genes in these regions that have mutations causative for CDH. Aim III: nonsynonomous SNPs identified will be studied as plausible causative mutations. Genome wide SNP analysis will be used to reveal regions of LOH in consanguineous Donnai Barrow kindreds and in discordant monozygotic twins. Aim IV: Candidate genes from animal models or from LOH regions with CDH in human will be studied for abnormal expression in human CDH lungs or diaphragms and tested in Drosophila or mouse cell-based or organ culture assays to determine functional significance. CDH gene defects uncovered by these studies will serve as targets for pharmacological or other therapeutic interventions to ameliorate or prevent this severe birth malformation.